Deck 15: Genetically Modified Organisms: Use in Basic and Applied Research

Question 1

When making a transgenic mouse by microinjection of foreign DNA into fertilized eggs

A) there is random integration of the transgene into the embryonic genome.
B) there is disruption or mutation of a targeted gene in the embryonic genome.
C) the resulting mice are genetically identical to the donor nucleus.
D) the integration event into the embryonic genome usually occurs by homologousrecombination.

Answer 1

there is random integration of the transgene into the embryonic genome.

Question 2

When making transgenic mice, to determine whether there has been stable integration of a transgene into the mouse chromosome, tail biopsies are taken from the mouse pups and tested for the presence of the transgene by

A) Northern blot
B) Southern blot
C) Western blot
D) Chromatin immunoprecipitation

Answer 2

Southern blot

Question 3

Mating mice from different founder lineages that carry identical transgenes will result in

A) one transgene being expressed and the other silenced.
B) a heterozygote for the transgene.
C) a true homozygote in which independent segregation of the transgenic loci is predictable.
D) a homozygote in which segregation of the transgenic loci is not necessarily independent.

Answer 3

a homozygote in which segregation of the transgenic loci is not necessarily independent.

Question 4

When making a mouse by gene targeting

A) there is random integration of the transgene into the embryonic genome.
B) there is disruption or mutation of a targeted gene in the embryonic genome.
C) the resulting mice are genetically identical to the donor nucleus.
D) the integration event into the embryonic genome usually occurs by nonhomologousrecombination.

Question 5

Assume you have transfected embryonic stem cells with a gene-targeting vector containing the neomycin resistance gene as a positive selection marker and the thymidine kinase gene as a negative selection marker. You observe that some cells are resistant to neomycin but killed by ganciclovir. What was the outcome of gene targeting procedure?

A) No recombination.
B) Homologous recombination.
C) Nonhomologous recombination.
D) The transfection did not work.

Question 6

You have received a lineage of "knockout mice" from a collaborator. What method would not be useful to confirm that the mice really do not express the targeted gene in all tissues of the body?

A) Northern blot
B) Neomycin sensitivity assay
C) Western blot
D) Reverse transcriptase-PCR

Question 7

Gene knockouts often result in embryonic lethality. What strategy do investigators often use to make it possible to study a gene's role later in development?

A) Gene knockout using an inducible tetracycline resistance gene.
B) Use of embryonic stem cells from a strain of mice with brown fur and a blastocyst from astrain of mice with black fur.
C) Implantation of embryos into a pseudopregnant female.
D) Inducible Cre/Lox system for site-specific recombination.

Question 8

Creation of which of the following does not rely on homologous recombination into the genome?

A) transgenic mice
B) knockout mice
C) knockin mice
D) conditional knockout mice

Question 9

Eduardo Kac the transgenic artist produced which of the following?

A) Mickey the GFP mouse
B) A sculpture of the first transgenic rat-sized mouse
C) Alba the GFP bunny
D) A photo montage of pronuclear microinjection

Question 10

Which of the following correctly orders cells according to their phenotypic potential from greatest to least?

A) pluripotent, totipotent, terminally differentiated
B) totipotent, terminally differentiated, pluripotent
C) terminally differentiated, pluripotent, totipotent
D) totipotent, pluripotent, terminally differentiated

Question 11

All of the following are true of stem cells except:

A) They can be found in many developing organs.
B) They are terminally differentiated.
C) They have a high replicative capacity.
D) They may differentiate to replace cells lost to injury.

Question 12

When cloning an organism by nuclear transfer

A) there is random integration of a transgene into the recipient cell's nuclear genome.
B) there is disruption or mutation of a targeted gene in the resulting organism.
C) the resulting organism is genetically identical to only the donor nucleus and not the recipient cell's nucleus.
D) the cytoplasm of the donor cell is transferred to the recipient cell.

Question 13

The term "reprogramming" refers to

A) genetic modification of cells for the purpose of reproductive cloning
B) altering gene expression to revert a somatic cell to a stem cell state
C) artificial telomere elongation to effectively reduce cell age
D) the inducible excision of a gene in a conditional knockout mouse

Question 14

The results of nuclear transplantation experiments in Xenopus laevis by John Gurdon and co-workers established the basic principle that

A) cell differentiation depends on changes in the expression not content of the genome.
B) cell differentiation depends on changes in the content not expression of the genome.
C) cell differentiation depends on changes in both the expression and content of the genome.
D) cloning by nuclear transfer is only possible in amphibians.

Question 15

When nuclear transfer is performed by cell fusion, the resulting cloned offspring

A) will be genetically identical to the original adult cell nucleus but the mitochondrial DNA willcome from the recipient egg.
B) will be genetically identical to the original adult cell nucleus but the mitochondrial DNA willcome from both the donor adult cell cytoplasm and the recipient egg.
C) will be genetically identical to the original adult cell nucleus and the mitochondrial DNA willcome from the donor adult cell cytoplasm.
D) will be a chimera of nuclear and mitochondrial DNA from the original adult celland the recipient egg.

Question 16

Which of the following is not a suspected reason for the inefficiency/poor outcomes of reproductive cloning?

A) inefficient reprogramming
B) premature terminal differentiation in the developing embryo
C) telomere length
D) chromosome missegregation during early embryonic divisions

Question 17

"Pharming" is

A) the growth of transgenic plants as food crops
B) the use of Agrobacterium tumefaciens to transform animal cells
C) the use of genetically modified organisms to produce pharmaceutical agents
D) the use of plants or algae to produce biofuels

Question 18

What information did the study of crown gall disease provide that was critical for plant genetic engineering?

A) Understanding the role of plant hormones in growth promotion.
B) Knowledge of plant tumor-inducing genes.
C) Understanding the role of opine synthesis enzymes in tumor progression.
D) Discovery of a plasmid that introduces virulence genes into plants.

Question 19

Plasmids are used as vectors in both plant and bacterial recombinant DNA technology. However, there is a major difference in the fate of genes introduced into bacteria on most bacterial plasmids and into plants on Ti plasmids. What is this difference?

A) In bacteria, genes are stably expressed; in plants, gene expression is quickly lost.
B) Gene expression tends to decrease rapidly and unpredictably in bacteria; gene expression is much more stable in plants.
C) Bacterial plasmids are circular DNAs; Ti plasmid DNA is linear.
D) Bacterial plasmids and the genes they carry usually are not integrated into the chromosome; Ti plasmids and the genes they carry are integrated into the chromosome.

Question 20

Which is not a method by which one can introduce foreign DNA into a plant cell?

A) calcium chloride treatment followed by heat shock
B) infection with a specific bacterium
C) microballistic "gene gun"
D) electroporation

Question 21

Describe the method for creating a transgenic mouse by pronuclear injection. What information can a transgenic mouse provide?

Question 22

You are trying to make a transgenic mouse but there is very poor survivorship of the embryos after introduction of the foreign gene by pronuclear injection. Describe a method you could use to get around this problem.

Question 23

You have cloned the cDNA for human XPA. You now attempt to use the human XPA cDNA (linked to a promoter) to correct the genetic defect in mice that have the mouse equivalent of the human disease xeroderma pigmentosum, by making transgenic mice. Briefly explain how you would analyze the transgenic mice for:
Stable integration of the transgene into the mouse's genomic DNA
Transcription of the transgene
Translation of the transgene
Ability of the XPA protein to correct the genetic defect in the transgenic mice.

Question 24

Describe the method for creating a knockout mouse by gene targeting. Explain the importance of selectable marker genes in this procedure. What information can a knockout mouse provide?

Question 25

You have created a strain of knockout mice. Describe an experiment to confirm that the mice lack expression of the targeted gene at the level of mRNA expression. Show sample positive results.

Question 26

Discuss some of the pros and cons of using genetically modified nonhuman primates in experimental medicine.

Question 27

Describe a recently developed method for creating transgenic livestock by linker-based sperm-mediated gene transfer. Why don't investigators just use pronuclear microinjection?

Question 28

Discuss some of the potential applications of "gene pharming."

Question 29

Describe amphibian nuclear transplantation experiments that established the basic principle that cell differentiation depends on changes in gene expression not changes in the content of the genome.

Question 30

Describe the method for cloning of mammals by nuclear transfer. What information can a cloned organism provide?

Question 31

Less than 1% of all nuclear transfers from adult differentiated cells result in normal-appearing cloned offspring. Give possible explanations for this lack of efficiency.

Question 32

Will a cloned calico cat have the exactly the same pattern of orange and black patches on her coat compare with her calico donor? Explain your answer.

Question 33

Discuss some of the controversial applications of cloning by nuclear transfer.

Question 34

Describe an experiment to determine whether the tomato you just bought from the grocery store is genetically modified. Show sample positive results.

Question 35

Design a Southern blot experiment to check a transgenic mouse's DNA for integration of the GFP cDNA under control of a constitutive promoter. You many assume any array of restriction sites you wish in the target mouse chromosome and in the GFP transgene.
(a) Show sample results for a successful and an unsuccessful integration.
(b) Will mice from different founder lineages have the same site of integration?
(c) Describe techniques you would use to analyze the transgenic mice for transcription of the transgene and translation of the transgene.

Question 36

What would be the outcome if the procedure used to make gene-targeted (knock-out) mice was altered in the following ways (consider each situation separately).
(a) The embryo-derived stem (ES) cells did not possess a gene responsible for visible phenotypic trait (such as agouti coat color) that differed from that of the cell in the recipient blastocyst.
(b) The inserted "disrupting" gene (e.g. neo^R) did not insert into the gene of interest.
(c) Chemical transfection or electroporation failed.
(d) The gene knock-out was embryonic lethal

Question 37

Consider the following hypothetical scenario:
A young scientific genius, Dolly, is terminally ill with hereditary nonpolyposis colon cancer (HNPCC). When she dies, her parents feel that one of the most remarkable minds in science will die with her, and they feel they owe it to the world to not let this happen. The family travels to a secret lab on a small offshore island, which allegedly performs "reproductive cloning"
(cloning by somatic cell nuclear transfer). Dolly's parents hope to clone her from one of her skin cells.
(a) If the lab is successful in cloning Dolly, can they guarantee that her clone will be as academically gifted as Dolly? Why or why not?
(b) Would Dolly and her clone have identical DNA "fingerprints"? Why or why not?
(c) Would Dolly and her clone share the same mitochondrial DNA? Why or why not?
(d) Would you expect there to be a difference in the length of telomeres in Dolly's skin cells versus in the cells of her embryonic clone? Why or why not?

Question 38

A team of molecular biologists seeks your advice regarding whether it would be more important to include regulatory elements such as insulators and matrix attachment regions in a gene construct for generating transgenic mice by gene-targeting in embryo-derived stem cells, or for generating transgenic mice by microinjection of foreign DNA into pronuclei of fertilized eggs? Provide advice and explain your rationale.